Radioactively labelled microspheres are commonly used to measure coronary blood flow. Decrease in microsphere content is known to occur in infarcted myocardium, introducing a potential source of error in flow measurement. To investigate stability of microsphere content in ischemic but noninfarcted muscle, we injected microspheres in 11 open-chest dogs and produced coronary occlusion. Four hours later microsphere content of ischemic endocardium fell to 78% of the value measured in normal endocardium. Such changes were not seen in ischemic epicardium. An additional 14 closed-chest dogs had preocclusion microspheres and sacrifice 3 days later. In these animals, microsphere content was decreased (to 55-67% of analogous normal zone values) approximately equally in ischemic but surviving muscle and in infarcted muscle. We conclude that microsphere "loss" may distort flow values even in relatively short experiments. Within 3 days microsphere "loss" occurs in both surviving and infarcting portions of myocardium in the region supplied by the occluded artery. This uniformity permits comparison of flow among various subdivisions of ischemic myocardium.